BACKGROUND/AIMS: Statins act as antineoplastic agents through the inhibition of cell proliferation. This study sought to demonstrate the effects of statins on extrahepatic bile duct cancer cell apoptosis and to document the changes in protein expression involved in tumor growth and suppression. METHODS: Human extrahepatic bile duct cancer cells were cultured. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were performed to determine the effect of statins on cell proliferation. Apoptosis was measured by a cell death detection enzyme-linked immunosorbent assay and caspase-3 activity assay, and flow cytometry was used to determine the percentage of cells in each phase of the cell cycle. The protein expression of Bax, Bcl-2, insulin-like growth factor 1 (IGF-1) receptor, extracellular signal-regulated kinase 1/2 (ERK1/2), and Akt was measured by Western blot analysis. RESULTS: Simvastatin suppressed cell proliferation by inducing G1 phase cell cycle arrest in bile duct cancer cells. Furthermore, it induced apoptosis via caspase-3 activation, downregulated the expression of the Bcl-2 protein, and enhanced the expression of the Bax protein. Moreover, simvastatin suppressed the expression of the IGF-1 receptor and IGF-1-induced ERK/Akt activation. CONCLUSIONS: Simvastatin induces apoptosis in bile duct cancer cells, which suggests that it could be an antineoplastic agent for bile duct cancer.
Antineoplastic Agents/pharmacology ; Apoptosis/*drug effects ; Bile Duct Neoplasms/*drug therapy ; Cell Cycle/drug effects ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Enzyme-Linked Immunosorbent Assay ; Flow Cytometry ; Humans ; Hypolipidemic Agents/*pharmacology ; Receptor, IGF Type 1/*drug effects ; Simvastatin/*pharmacology

OBJECTIVETo investigate the therapeutic value of inhibiting the expression of insulin-like growth factor-I receptor (IGF-IR) using picropodophyllin (PPP) by studying the effects on proliferative and metastatic potentials of human hepatocellular carcinoma (HCC) using an in vitro cultured cell system.

METHODSIGF-IR expression in human HCC cell lines (Bel-7404, Bel-7402, HepG2, and Huh-7) and human hepatocytes (L02) was assessed at baseline (pre-treatment) and after PPP treatment by western blotting. Changes in cell cycle were analyzed by flow cytometry and in cell viability by sulforhodamine B staining. Early apoptosis was detected by annexin-V/FITC and propidium iodide double-staining assay. Caspase-3/7 activity was suppressed by z-VAD-FMK and analyzed by homogeneous luminescence assay. Effects on cell motility were tested by wound-scratch test. Between-group differences were assessed by t-test or one-way analysis of variance.

RESULTSIGF-IR was markedly up-regulated in all HCC cell lines (vs. non-hepatoma hepatocytes). HCC cells with PPP-inhibited IGF-IR showed time-dependent decreases in cell motility and viability. After treatment with PPP for 24 hours, the proportion of HCC cells in G1 phase was 2.1% +/- 0.4%, in S phase was 11.0% +/- 0.7%, and in G2/M phase was 87.1% +/- 0.6%, and no healing was observed in the wound-scratch assay. The PPP treatment induced cell apoptosis, as evidenced by enhanced caspase-3/7 activity; the proportion of annexin-V+/PI- cells was significantly higher in the HepG2 cells than in the non-hepatoma hepatocytes (16.4% +/- 0.4% vs. 5.8% +/- 0.2%, t = 14.05, P less than 0.01). After z-VAD-FMK treatment, the apoptosis rate was significantly higher in the HepG2 cells than in the non-hepatoma hepatocytes (11.3% +/- 0.7% vs. 5.8% +/- 0.2%, t = 11.83, P less than 0.01).

CONCLUSIONIGF-IR is associated with proliferation, cell motility, and apoptosis of HCC cells, and may be a promising molecular target for HCC.

Apoptosis ; drug effects ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Podophyllotoxin ; analogs & derivatives ; pharmacology ; Receptor, IGF Type 1 ; metabolism

This study was aimed to explore the effects of insulin on expression of insulin receptor (IR) and insulin-like growth factor-I receptor (IGF-IR) in Reh cells and promoting effect on proliferation of Reh cells. The proliferation of Reh cells were evaluated by CCK-8 assay. The expression levels of IR and IGF-IR mRNA in Reh cells at different times were detected by real-time quantitative polymerase chain reaction. The results showed that insulin promoted the proliferation of Reh cells in dose- and time-dependent manners. Compared with the control group, insulin promoted the proliferation of Reh cells obviously (P < 0.05). When Reh cells were treated with insulin 10(-9) mol/L for 24, 48 and 72 h, the relative quantity of IR expression (2(-ΔCt1)/2(-ΔCt2)) was 2.2520 ± 0.7431, 1.9956 ± 0.9692 and 3.9766 ± 1.3189, respectively, the relative quantity of IGF-IR expression was 1.0803 ± 0.2238, 1.6026 ± 0.6158 and 3.1013 ± 0.1008, respectively, compared with the control group. The expression levels of IR and IGF-IR mRNA in Reh cells treated with insulin were obviously increased compared with the control group. It is concluded that insulin promotes the proliferation of Reh cells. The high expression levels of IR and IGF-IR may closely related with the growth of leukemia cells.
Cell Line, Tumor ; Cell Proliferation ; drug effects ; Humans ; Insulin ; pharmacology ; Leukemia, Lymphoid ; metabolism ; pathology ; Receptor, IGF Type 1 ; metabolism ; Receptor, Insulin ; metabolism

As a hormone with a number of biological effects, insulin not only displays the function of classic metabolic regulation, but also can regulate cell proliferation and differentiation, and ensure growth and development of embryos and young individuals. In vitro insulin can stimulate cell proliferation and differentiation. Insulin is also an important growth regulator in vivo, which has been proved in more and more studies. The role of insulin at the cellular level is triggered by the binding of insulin to its receptor located in the cell surface. However, insulin at the higher concentration can also been triggered by insulin-like growth factor-1 (IGF-1) receptor. Its role varies in different cell lines. Insulin receptor and insulin-like growth factor receptor-1 are widely expressed in human MDS and AML cell membranes. Recently, many studies related to the relationship between hyperinsulinemia and cancer have been reported. In this review the role and its possible mechanism in promoting human leukemia cell proliferation and inhibiting human leukemia cell proliferation are summarized. Furthermore, the potential application prospect of insulin analogues also will be described.
Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Humans ; Insulin ; pharmacology ; Leukemia ; metabolism ; pathology ; Receptor, IGF Type 1 ; metabolism ; Receptor, Insulin ; metabolism

Silibinin, from milk thistle (Silybum marianum), is a flavonolignan with anti-oxidative and anti-inflammatory properties. It has been therapeutically used for the treatment of hepatic diseases in China, Germany and Japan. Recently, increasing evidences prove that silibinin is also a potent antitumor agent, and the major anti-tumor mechanism for silibinin is the prominent inhibition of the activities of receptor tyrosine kinases (RTKs) and their downstream signal molecules in a variety of tumor cell lines, such as epidermal growth factor receptor 1 (EGFR) and insulin-like growth factor 1 receptor (IGF-1R) signaling pathways. Meanwhile, the evidences that silibinin selectively scavenges hydroxyl free radical (*OH) and specifically inhibits the action of nuclear factor kappaB (NF-kappaB) provide more complicated explanations for its antioxidant and anti-inflammatory effects. Some new findings such as that silibinin attenuating the cognitive deficits induced by amyloid beta protein (Abeta) peptide through its antioxidative and anti-inflammatory properties is valuable to broad the medical prospect of silibinin. In this review, we discuss the molecular pharmacological mechanisms of silibinin, focusing on its inhibition of tyrosine kinases, actions of antioxidation, free radical scavenging, immunoregulation and anti-inflammation.
Amyloid beta-Peptides ; metabolism ; Animals ; Anti-Inflammatory Agents ; pharmacology ; Antineoplastic Agents, Phytogenic ; pharmacology ; Antioxidants ; pharmacology ; Enzyme Activation ; Free Radical Scavengers ; pharmacology ; Humans ; Milk Thistle ; chemistry ; Molecular Structure ; NF-kappa B ; metabolism ; Protein-Tyrosine Kinases ; metabolism ; Reactive Oxygen Species ; metabolism ; Receptor Protein-Tyrosine Kinases ; metabolism ; Receptor, Epidermal Growth Factor ; metabolism ; Receptor, IGF Type 1 ; metabolism ; Signal Transduction ; drug effects ; Silymarin ; chemistry ; isolation & purification ; pharmacology

OBJECTIVETo establish a cetuximab-resistant human nasopharyngeal carcinoma 5-8F/Erbitux cell line and preliminarily study the relationship between the insulin-like growth factor 1 receptor (IGF-1R) signaling pathway and the resistance of nasopharyngeal carcinoma to cetuximab.

METHODSA nasopharyngeal cancer cell line, 5-8F, with high epidermal growth factor receptor (EGFR) expression and cetuximab sensitivity, was selected as study object. The cetuximab-resistant 5-8F/Erbitux cell line was induced by stepwise selection after exposure to increasing doses of cetuximab. The IC(50) was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the resistance index (RI) was calculated. The growth curves of 5-8F and 5-8F/Erbitux cells were plotted and the doubling times were calculated by cell counting assay. The cell cycle was detected by flow cytometry. Cross-resistance profiles of 5-8F/Erbitux cells to 5-Fu, Taxol and DDP were tested by MTT assay. Expression levels of P-gP, IGF-1R and P-IGF-1R of 5-8F and 5-8F/Erbitux cells were determined by Western blot analysis and MDR1 gene by real-time fluorescent quantitative PCR.

RESULTSA cetuximab-resistant human nasopharyngeal carcinoma cell line 5-8F/Erbitux was successfully established and their resistance index (RI) were 1.2 and 1.1, respectively, at 3 d and 5 d of the cetuximab treatment. The doubling times of 5-8F and 5-8F/Erbitux cells were 26.63 h and 142.30 h, respectively. Flow cytometry demonstrated that 5-8F/Erbitux cells showed an increased population at G(0)/G(1) phase (P < 0.001) and reduced population at S phase (P < 0.001), compared with 5-8F cells. The 5-8F/Erbitux cells showed cross-resistance to 5-Fu (RI = 3.95, P < 0.01) and some resistance to Taxol as well as enhanced sensitivity to DDP (P > 0.05 for all). The 5-8F/Erbitux cells also had increased levels of P-gP, IGF-1R and P-IGF-1R compared with 5-8F cells (P < 0.001 for all). Expression of MDR1 gene was not detected in 5-8F cells and only very weak expression in 5-8F/Erbitux cells.

CONCLUSIONCetuximab-resistant 5-8F/Erbitux cells have no common multidrug resistance like that induced by traditional chemotherapeutic drugs. The excessive activation of the IGF-1R signaling pathway is probably one of the mechanisms that caused resistance of 5-8F/Erbitux cells to cetuximab.

ATP-Binding Cassette, Sub-Family B, Member 1 ; metabolism ; Antibodies, Monoclonal ; pharmacology ; Antibodies, Monoclonal, Humanized ; Antimetabolites, Antineoplastic ; pharmacology ; Antineoplastic Agents ; pharmacology ; Antineoplastic Agents, Phytogenic ; pharmacology ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cetuximab ; Cisplatin ; pharmacology ; Drug Resistance, Neoplasm ; Fluorouracil ; pharmacology ; Humans ; Nasopharyngeal Neoplasms ; metabolism ; pathology ; Paclitaxel ; pharmacology ; Phosphorylation ; Receptor, IGF Type 1 ; metabolism ; Signal Transduction

OBJECTIVETo investigate the effects and the mechanisms of stanozolol (ST) on the proliferation, maturation and differentiation of in vitro cultured growth plate chondrocyte isolated from gonadotropin releasing hormone analogue (GnRHa)-treated adolescent rats, to study if ST mediates the proliferation of chondrocytes via the estrogen receptor alpha (ERalpha), androgen receptor (AR) and/or insulin-like growth factor-1 receptor (IGF-1R) and interactions of the two receptor and IGF-1R receptor signaling pathway, to investigate the mechanism of the biological effects in ST promoting bone growth/maturity at molecular level.

METHODThe rats were weaned at the end of 3 weeks and intramuscular injection of triptorelin of GnRHa preparations, qow x 2 was started. The rats were sacrificed at the end of 7 weeks, and then the tibiae growth plates were taken out with sterile procedure. The chondrocytes were obtained by two-time enzyme digestion method, and the experiments were carried out with the primary chondrocytes. Immunohistochemical staining of proliferating cell nuclear antigen (PCNA) and Western blot analysis were applied.

RESULTThe results of PCNA demonstrated that stanozolol enhanced the proliferation of the chondrocytes, time-course studies showed that the proliferation were maximally stimulated by stanozolol after 2 days of incubation and decreased again after longer periods of incubation. The expression of p-ERalpha, p-IGF-1R and p-extracellular-signal regulated kinase 1/2 (ERK1/2) increased with the incubation period of ST treatment, and reached the peak value at a certain time, and then gradually decreased. The expression of p-ERalpha, p-IGF-1R and p-ERK1/2 increased with the elevation of ST concentration, and reached the peak value at 10(-9) - 10(-8) mol/L, then gradually decreased. ST induced-p-ERalpha expression was partially blocked by ERalpha and mitogen-activated protein kinase kinase inhibitors. ST induced-p-IGF-1R expression was partially blocked by ERalpha and IGF-1R inhibitors. ST induced-p-ERK1/2 expression was partially blocked by mitogen-activated protein kinase kinase and IGF-1R inhibitors.

CONCLUSIONAs an androgen derivation, ST exerts its biological effects of promoting proliferation of the long bone growth plate chondrocytes via activating the classic ERalpha receptor pathway and mitogen-activated protein kinase pathway, and at the same time, by activation of IGF-1R. Both IGF-1R and ERalpha can promote "cross-talk" of two systems' receptor signal through mitogen-activated protein kinase signal pathway.

Androgens ; pharmacology ; Animals ; Cells, Cultured ; Chondrocytes ; cytology ; drug effects ; metabolism ; Estrogen Receptor alpha ; metabolism ; Female ; Growth Plate ; drug effects ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Rats ; Receptor Cross-Talk ; Receptor, IGF Type 1 ; metabolism ; Signal Transduction ; drug effects ; Stanozolol ; pharmacology

OBJECTIVETo investigate the effect of Chinese herbs for nourishing yin and removing fire (NYRF) on gene expressions of estrogen receptor alpha (ER alpha), insulin-like growth factor-1 receptor (IGF-1R) and epithelial growth factor receptor (EGFR) in the epiphyseal growth plate of the female pubertal rats.

METHODSThe rats were randomly divided into the control group and the intervened group. Immunohistochemistry and realtime-PCR methods were used to measure the gene expression of ER alpha, IGF-1R and EGFR and their protein synthesis in epiphyseal growth plate.

RESULTSAfter being intervened with NYRF, the gene expressions of ER alpha and IGF-1R were down-regulated and their protein synthesis markedly reduced, while those of EGFR were unchanged.

CONCLUSIONNYRF can modulate the development and maturation of bone by regulating the expressions of ER alpha and IGF-1R in the epiphyseal growth plate.

Animals ; Disease Models, Animal ; Drugs, Chinese Herbal ; pharmacology ; Estrogen Receptor alpha ; genetics ; metabolism ; Female ; Gene Expression ; drug effects ; Growth Plate ; drug effects ; metabolism ; Humans ; Protein Biosynthesis ; drug effects ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Receptor, Epidermal Growth Factor ; genetics ; metabolism ; Receptor, IGF Type 1 ; genetics ; metabolism ; Yin-Yang

The importance of insulin-like growth factor 1 receptor (IGF-1R) signaling in malignant behaviour of tumour cells is well established. Inhibiting the activity of IGF-1R may result in striking apoptosis in malignant cells growing. IGF-1R antibodies which are currently in phase I and II clinical trials and several IGF-IR TKIs have preclinically been characterized. This review describes recent developments of small molecule tyrosine kinase inhibitors.
Animals ; Apoptosis ; drug effects ; Benzimidazoles ; pharmacology ; Catechin ; analogs & derivatives ; pharmacology ; Cell Line, Tumor ; Humans ; Neoplasms ; pathology ; Piperazines ; pharmacology ; Protein Kinase Inhibitors ; pharmacology ; Pyridones ; pharmacology ; Pyrimidines ; pharmacology ; Pyrroles ; pharmacology ; Receptor, IGF Type 1 ; antagonists & inhibitors ; chemistry ; metabolism ; Signal Transduction

OBJECTIVETo evaluate the effect of insulin-like growth factor (IGF) and its receptor-I antibody on the growth of human adrenocortical carcinoma SW-13 cell line in vitro.

METHODThe growth curves of SW-13 cell treated with IGF and its receptor-I antibody were obtained by means of MTT assay. The effects of the two agents, added either alone or in combination at different concentrations, on the cell growth were evaluated.

RESULTSIGF significantly promoted proliferation of SW-13 cells, and its effect was positively correlated with its concentrations (P<0.05). IGF receptor-I antibody inhibited the effect of insulin-like growth factor with direct inhibitory effect on proliferation of SW-13 cells (P<0.05).

CONCLUSIONIGF can promote the growth of human adrenocortical carcinoma SW-13 cells via its receptor-I. IGF receptor-I antibody can inhibit the effect of the growth factor, suggesting a possible role of this receptor in the treatment of adrenocortical carcinoma.

Adrenocortical Carcinoma ; pathology ; Antibodies ; pharmacology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Dose-Response Relationship, Drug ; Humans ; Insulin-Like Growth Factor I ; pharmacology ; Receptor, IGF Type 1 ; immunology